It was confirmed by polyacrylamide gel electrophoresis that isolated 16S rRNA was cleaved by the active component (protein A) or the active fragment (T2A) of colicin E3. However, the degradation was random, in contrast with the specific cleavage observed in the interaction of colicin E3 with ribosomes. Furthermore, the active component and the active fragment had low activities, and far greater amounts of these materials were required for degradation of the isolated rRNA than for ribosome inactivation. The degradation of rRNA cannot be due to contaminating ribonuclease(s), but is due to colicin E3 itself, because of the following facts. (1) Protein B of colicin E3, which specifically inhibits the ribosome-inactivating activity of colicin E3, inhibited the degradation of rRNA. (2) Protein B of colicin E2, which inhibits the action of colicin E2 but not of colicin E3, failed to inhibit the degradation of rRNA. (3) The activity appeared in the peak of protein A or fragment T2A, respectively, when they were rechromatographed on Sephadex G-75.